We report the presence of coenzyme factor 430 (F430), a prosthetic group of methyl coenzyme M reductase for archaeal methanogenesis, in the deep sub-seafloor biosphere. At 106.7m depth in sediment collected off Shimokita Peninsula, northwestern Pacific, its concentration was estimated to be at least 40fmolgsediment −1 (i.e. 36pgg −1 wet sediment). This is about three orders of magnitude lower than typical concentrations of archaeal intact polar lipids in similar sub-seafloor sediments. On the basis of the concentration of F430 in methanogens and conversion to biomass composed of typical sub-seafloor microbial cells, we estimated that ca. 2×10 6 cellsg −1 could be methanogens in the deeply buried marine sediment.